Covalent attachment of photoremoveable group to a parent compound (i.e. "caging") to alter its physical or biological properties has been exploited extensively for following components of dynamic systems. The term "cage" refers to a photolytically sensitive substituent that is designed to interfere with the reactivity or other physical properties of the free probe. Photolysis (typically by illumination in the UV (250-400 nm) region of the spectrum) cleaves off the caging group, restoring the normal properties of the parent compound. In this way it is possible to release the parent compound into the system of interest with much better temporal and spatial resolution than is possible by simple diffusion.
In the use of caged compounds for the study of processes that change rapidly, such as biological processes that occur on the time scale of a millisecond or faster, it is critical that photolysis also occur very rapidly and with relatively high quantum yield. Also of critical importance is that the caged compound is stable and still soluble in the system of interest, and that caging reduces some property of the parent compound to the desired level. The o-nitrobenzyl group and its substituted variants have been widely used to prepare caged versions of biologically active substances by attachment at a variety of terminal substituents, including e.g. aliphatic hydroxyls (J. Chem. Soc. Perkin Trans. 1,2161 (1993)); phenolic hydroxyls (Biochem. 32, 1338 (1993)); amines (caged as o-nitrobenzylamines (Biochem. 33, 1526 (1994)) and also as o-nitrobenzylcarbamates (J. Physiol. (London) 465, 1 (1993)); amides (caged on the nitrogen (J. Org. Chem. 58, 4599 (1993)), carbamates (caged on the nitrogen (Biochem. 28, 49 (1989)), phosphates (J. Am. Chem. Soc. 110, 7170 (1988)); and carboxylates (caged as o-nitrobenzyl esters) (J. Org. Chem. 55, 1585 (1990)). Unfortunately, the photolysis properties have varied widely with both the caged compound and the substitution pattern on the o-nitrobenzyl caging group, i.e. photolysis rates from &lt;1 s.sup.-1 up to 10.sup.5 s.sup.-1, and quantum yields ranging from &lt;0.01 up to 0.8, and efforts at preparing caged materials with the characteristics desired for use in biological systems have met with limited success.
For example, many biologically active materials, e.g. neurotransmitters, peptides, proteins, hormones, second messengers, contain carboxylic acid moeities. Carboxylates caged as their o-nitrobenzyl esters, however, photolyze only with low quantum yield (&lt;0.01) and at a very low rate (ca. 1 s.sup.-1) (11). Mother problem has been that caging of carboxylates as their o-nitrobenzyl esters significantly dishes their solubility in aqueous systems. A recently reported caging group for the carboxylate function of glycine (2-methoxy-5-nitrophenoxy ester) is photolyzed in the microsecond time region with a 0.2 quantum yield but is hydrolyrically unstable and thus not useful for biological applications (12). Some functionalities have been caged with the .alpha.-carboxy-o-nitrobenzyl substituent, i.e. phenols (Biochem. 32, 1338 (1993)), amines (Biochem. 33, 1526 (1994)), amides (J. Org. Chem. 58, 4599 (1993)), and carbamates (Biochem. 28, 49 (1989)), with generally favorable results; yet a comparison of caged compounds that were prepared by attaching a substituted or unsubstituted o-nitrobenzyl group (including N-.alpha.-carboxy-o-nitrobenzyl) at the terminal amine of the neurotransmitter glutamate showed mixed results (Table 1; Compounds A, B, C). Utilizing this comparison, the .gamma.-(.alpha.-methyl-o-nitrobenzyl) ester of glutamate (D) was prepared; however, its photolysis rate (9 s.sup.-1) and quantum yield (very low) were unacceptable (PNAS/USA 91 (19), 8752 (1994)). In contrast to the previous results, however, attachment of an .alpha.-carboxyl substituent to the .gamma.-(o-nitrobenzyl) ester of glutamate unexpectedly provided a water-stable compound (3) with dramatically improved photolysis rate and quantum yield, several orders of magnitude over previously prepared versions of o-nitrobenzyl-caged carboxylates (J. Org. Chem. 55, 1585 (1990)). This caging group can be used to prepare caged versions of a number of biologically useful acid derivatives. Furthermore, attachment of this .alpha.-carboxy-o-nitrobenzyl ester to carboxylates maintains the aqueous solubility that has normally been lost in the past by converting carboxylates into their o-nitrobenzyl esters.
TABLE 1 ______________________________________ Relative rates of photolysis for related caged compounds. ##STR1## ##STR2## k (s.sup.-1) Compound at 308 nm .PHI. ______________________________________ A 210 &lt;0.01 B 385 0.04 C 2,240 0.06 D 9 &lt;0.01 3 24,800 0.16 ______________________________________